The objective of the experiments described in this proposal is to identify, isolate, and determine aspects of the molecular structure of the beta-adrenergic receptor. This is an initial and necessary first step in order to determine the mechanism by which catecholamines activate adenylate cyclase in the plasma membrane of responsive cells. Work will be performed on the beta-receptor-adenylate cyclase system of the nucleated avian erythrocyte ghost and various cloned cell lines. Several methods will be used to isolate the specific catecholamine receptor, using chemically stable derivatives of beta-receptor antagonists. These methods are: (a) specific derivatization of the receptor using photo-affinity labels; (b) purification of the detergent-solubilized receptor using ion exchange and molecular sieve columns; (c) affinity chromatograppy using agarose-bound avidin-biotinyl-beta-ligand complexes and agarose-bound anti-DNP-DNP-beta-ligand complexes; (d) surface distribution of catecholamine receptor on cloned cell lines and avian erythrocytes; (e) immunoprecipitation of the receptor from detergent extracts of plasma membrane using dinitrophenyl (DNP) or biotinyl derivatives of receptor antagonists and anti-DNP antibody or avidin; (f) ultracentrifugation of detergent-solubilized receptor-DNP ligand complexes or receptor-biotinyl ligand complexes with Ferritin-conjugated anti-DNP antibody or avidin. The molecular size, subunit structure, and molecular composition of the receptor will be determined. Antibody to the receptor will be prepared for the purpose of using Ferritin conjugates to determine receptor distribution on the surface of responsive cells. The experiments will increase our understanding of catecholamine beta-receptors, which function in controlling heart rate, the metabolic state of liver and muscle, and autonomic functions.